Neurotensin receptor agonists and opioid receptor agonists

ABSTRACT

This document provides methods and materials for treating pain. For example, this document provides methods that involve administering a neurotensin receptor (NTR) agonist and an opioid receptor agonist to a mammal (e.g., a human). Compositions containing an NTR agonist in combination with an opioid receptor agonist also are provided.

1. REFERENCE TO RELATED APPLICATIONS

This is a continuation of U.S. application Ser. No. 12/714,476, filedFeb. 27, 2010, which is a divisional of U.S. application Ser. No.11/709,991, filed Feb. 23, 2007, which claims priority to U.S.Provisional Application Ser. No. 60/776,248 filed Feb. 24, 2006,entitled “Neurotensin Receptor Agonists and Opioid Receptor Agonists,”and U.S. Provisional Application Ser. No. 60/785,233, filed Mar. 22,2006, entitled “Synergistic Neurotensin-Opiate Compositions and Methodsof Use,” all of which are hereby expressly incorporated by reference intheir entirety.

BACKGROUND

2. Technical Field

The subject matter herein relates to analgesic compositions,formulations, and methods of use and to synergistic combinations ofneurotensins or analogs and opiates or analogs that reduce their sideeffect profile at the same or increased analgesic potency.

3. Background Information

Analgesics are used in the treatment of pain, the cause of which canvary from acute wounds such as cuts, bruises, surgical incisions, orburns to chronic conditions such as structural defects (e.g., back,joint, or spinal disc problems) or diseases (e.g., cancer, inflammatoryconditions, or infections).

Analgesics have an ability to reduce perception of pain impulses by thecentral nervous system. Opiates are the most widely used analgesics inthe treatment of severe pain. Opiates bind a variety of receptors,including mu, delta, and kappa receptors. Both the endogenous opioidpeptides and synthetic opiate analgesics alter the central or peripheralrelease of neurotransmitters from afferent nerves sensitive to noxiousstimuli. The specific actions of the narcotic analgesics can be definedby their sensitivity and selectivity for binding at three specificopiate receptor types, mu, kappa, and delta. The mu opiates have highaffinity and selectivity for binding sites in the brain and analgesicaction is mostly attributed to these receptors. Delta receptors aremostly located in the spinal cord and may have a role in peripheralpain. Kappa receptors are located in the hypothalamus and may accountfor the neuro-endocrine actions associated with kappa binding.

Narcotic analgesics are also classified as agonists, mixedagonist-antagonists, or partial agonists by their activity at opiatereceptors. Narcotic agonists include natural opium alkaloids (e.g.,morphine, codeine), semisynthetic analogs (e.g., hydromorphone,oxymorphone, oxycodone), and synthetic compounds (e.g., meperidine,levorphanol, methadone, sufentanil, alfentanil, fentanyl, remifentanil,levomethadyl). Mixed agonist-antagonist drugs (e.g., nalbuphine,pentazocine) have agonist activity at some receptors and antagonistactivity at other receptors; partial agonists (e.g., butorphanol,buprenorphine) are also included. Narcotic antagonists (e.g., naloxone)do not have agonist activity at any of the opiate receptor sites.Antagonists block the opiate receptor, inhibit pharmacological activityof the agonist, and precipitate withdrawal in dependent patients.

When given acutely in the treatment of severe pain, e.g., in post-oppain, opiates produce a variety of secondary pharmacologicalside-effects, ranging from mild to life threatening. Cough andrespiration are depressed, and fatal doses lead to severe respiratorydepression by direct inhibition of the respiratory center in the brainstem. Nausea and vomiting occur in many individuals through directstimulation of the chemoreceptor trigger zone. Therapeutic doses alsoresult in inhibition of baroreceptor responses and hypotension, thelatter through the release of histamine. Gastrointestinal motility isreduced, resulting in constipation. Sedation occurs and cognitivefunction is impaired. Extended use of opiates, as in the treatment ofcancer pain, is associated with dependence, tolerance, and potential fordrug abuse.

Neurotensin and its analogs are also potent analgesic in animals. Likeopiates, they are produced in the brain, spinal cord dorsal horn,hypothalamus, and gut. In all these locations, cells producingneurotensin are in close proximity to those producing endogenousopiates, which is consistent with the fact that neurotensin and opiateshave similar actions. Several different neurotensin receptors (NTRs,e.g., NTR1, NTR2, and NTR3) have been identified to date, presumablywith slightly different functions. Several similarities exist in theactions between neurotensin and opiates. First, neurotensin receptorsinvolved in the treatment of central pain may be different than thoseinvolved in the treatment of peripheral pain. Second, neurotensinadministration is associated with not just analgesia but hypotension(unrelated to histamine release), fall in basal temperature, and weightloss. Third, neurotensin induces tolerance. However, unlike opiates,neurotensin does not depress respiration, suppress coughing, induceconstipation, alter cognitive function or cause sedation. Neurotensin isknown to increase gastrointestinal transit and induce diarrhea.Neurotensin has also been shown to exhibit antipsychotic effect andantiparkinsonian effect.

To minimize side effects generated by giving a specific opiate,different opiates can be combined to produce synergistic analgesiceffects. Synergism is defined as correlated action of two or more agentsso that the combined action is greater than the sum of each actingseparately. For example, when morphine and methadone are combined,analgesic synergy is achieved but not accompanied by synergistic effectin other pharmacological actions, such as in gut motility. This synergybetween the opiates is useful because unwanted side effects associatedwith both acute and long-term administration of the opiates can bediminished without reducing analgesic potency. However, synergisticaction between different opiates remains unclear because two opiatesacting on the same receptors, namely mu receptors, may not exhibitsynergy. For example, methadone is only synergistic with morphine,codeine, 6-acetyl morphine, and morphine-6-beta glucuronide but not withfentanyl, oxymorphone, oxycodone, meperidine, or alfentanyl. All of theabove mentioned opiates are mu receptor agonists. Similarly, morphine issynergistic only with methadone but none of the other mu agonists.

Opiates remain the drug of choice in management of severe pain to date.However, new agents and methods are needed to provide alternative painmanagement, with or without opiates. Alternative agents and methods areneeded to enhance the pharmacological effect and minimize unwanted sideeffects of opiates, such as tolerance, dependence, and constipation.

SUMMARY

The subject matter herein provides compositions and methods for treatingpain by combining different analgesics to achieve synergy between theanalgesic agents and reduce their side effect profile. Although themechanism of action and receptor binding for different classes ofanalgesics are different, the results provided herein demonstrate thatthe use of opiates or opiate receptor agonists in combination with otheranalgesics, such as neurotensins or neurotensins receptor agonists, canachieve a synergistic analgesic effect and can reduce constipation aswell as tolerance and dependence to both opiate and NT. The method bywhich opiate dependence is diminished or blocked is not just thatsmaller doses of opiate can be used but also that NT can block thedopaminergic reward system. Furthermore, the hypotension attributable tolarger doses of NT can be eliminated.

NTR agonists and opioid receptor agonists are typically administered inamounts effective to reduce the level of pain experienced by the mammal.As disclosed herein, administering an NTR agonist together with anopioid receptor agonist provides a mammal with a greater level of painrelief than when either the NTR agonist or the opioid receptor agonistis used alone. Examples of NTR agonists include, without limitation,neurotensin (NT) polypeptide analogs such as NT69L. Examples of opioidreceptor agonists include, without limitation, morphine, codeine, andnalorphine hydrochloride. The subject matter herein also providescompositions containing an NTR agonist in combination with an opioidreceptor agonist. For example, a composition can be formulated tocontain morphine and NT69L. The compositions provided herein can be usedto treat pain.

In one aspect, the subject matter herein features methods for treatingpain comprising administering a neurotensin receptor agonist and anopioid receptor agonist to a mammal. The neurotensin receptor agonistcan be a polypeptide. The polypeptide can contain an amino acid analog(e.g., L-neo-Trp). The polypeptide can be selected from the groupconsisting of NT(1-13), NT(8-13), NT69L, NT69L′, and NT76. Theadministering can be by injection. The opioid receptor agonist can bemorphine. The method can comprise administering a composition containingthe neurotensin receptor agonist and the opioid receptor agonist. Themethod can comprise administering the neurotensin receptor agonist priorto administering the opioid receptor agonist. The method can compriseadministering the opioid receptor agonist prior to administering theneurotensin receptor agonist.

In another aspect, the subject matter herein features a compositioncontaining a neurotensin receptor agonist and an opioid receptoragonist. The neurotensin receptor agonist can be a polypeptide selectedfrom the group consisting of NT(1-13), NT(8-13), NT69L, NT69L′, andNT76. The opioid receptor agonist compound can be morphine. Thecomposition can contain a pharmaceutically acceptable carrier. Thecomposition can contain two or more neurotensin receptor agonists. Thecomposition can contain two or more opioid receptor agonists.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Although methods and materialssimilar or equivalent to those described herein can be used to practicethe invention, suitable methods and materials are described below. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph plotting the percent maximum possible effect at theindicated time post injection for rats treated with morphine alone (2.5or 10.0 mg/kg), NT69L alone (0.5 mg/kg), or both morphine (2.5 mg/kg)and NT69L (0.5 mg/kg).

FIG. 2 is a graph plotting the percent maximum possible effect at theindicated time post injection for rats treated with morphine alone (5.0mg/kg), NT77 alone (10.0 mg/kg), NT72 alone (2.5 mg/kg), both morphine(5.0 mg/kg) and NT77 (10.0 mg/kg), or both morphine (5.0 mg/kg) and NT72(2.5 mg/kg).

DETAILED DESCRIPTION

The methods described herein include administering at least one (e.g.,one, two, three, four, five, six, or more) NTR agonist and at least one(e.g., one, two, three, four, five, six, or more) opioid receptoragonist to a mammal (e.g., a mouse, rat, dog, cat, horse, cow, pig,monkey, or human). The term “NTR agonist” as used herein refers to anymolecule that binds to an NTR and induces an NTR response. NTR agonistsinclude, without limitation, polypeptides and other agents such as smallmolecules. For example, an NTR agonist can be a polypeptide such as NT,NT(1-13), NT(8-13), and NT69L. Examples of opioid receptor agonistsinclude, without limitation, alfentanil hydrochloride, alphaprodine,anileridine hydrochloride, buprenorphine hydrochloride, butorphanoltartrate, codeine, codeine phosphate, codeine sulfate, fentanyl citrate,heroin, hydrocodone tartrate, hydromorphone hydrochloride, ketobemidone,levorphanol tartrate, meperidine hydrochloride, meptazinolhydrochloride, methylfentanyl, morphine, morphine acetate, morphinesulfate, nalbuphine hydrochloride, nalorphine hydrochloride, oxycodonehydrochloride, oxymorphone hydrochloride, pholcodine, remifentanilhydrochloride, sufentanil citrate, and tramadol hydrochloride.

Typically, NTR agonists induce NTR responses such as antinociception,hypothermia, diminished food consumption, blockade of muscle rigidity(catalepsy) caused by antipsychotic drugs (e.g., haloperidol), andinhibition of climbing behavior caused by the dopamine receptor agonistapomorphine. NTR responses can be measured using any method. Forexample, antinociception can be measured using pain tests such as tailflick and paw withdrawal studies. Briefly, tail flick and paw withdrawalstudies typically involve subjecting an animal to a painful stimulus(e.g., heat, a pin prick, or a pinch on the foot), and measuring thelength of time or amount of pinching force applied before the animalphysically responds to the stimulus by flicking its tail or withdrawingits paw. NTR effects also can be measured in any suitable cell system.For example, NTR effects (at NTR1) can be measured in human colonicadenoma cells (HT29 cells) by measuring the formation of secondmessengers (e.g., release of inositol phosphates or increase inintracellular levels of calcium ions). The specificity of NTR responsescan be confirmed using NTR antagonists such as SR48692 and SR142948A.

NT is a tridecapeptide (Carraway and Leeman (1973) J. Biol. Chem.248:6854-6861) that induces antinociception and hypothermia upon directadministration to brain. Systemic administration of NT does not inducethese effects, however, since NT is rapidly degraded by proteases andhas poor blood brain barrier permeability. NT behaves as aneurotransmitter or neuromodulator in the CNS, and there are strikinginteractions between NT (via its receptors) and central dopaminergicsystems (Tyler-McMahon et al. (2000) Regul. Pept. 93:125-136; andLambert et al. (1995) Ann. NY Acad. Sci. 757:377-389).

The complete amino acid sequence of NT(1-13) ispyroGlu-Leu-Tyr-Glu-Asn-Lys-Pro-Arg-Arg-Pro-Tyr-Ile-Leu (SEQ ID NO:1).Most, if not all, of the activity mediated by NT(1-13) also can be seenwith the shorter fragment, NT(8-13), which has the sequenceArg-Arg-Pro-Tyr-Ile-Leu (SEQ ID NO:2). These NT polypeptides, as well asother NTR agonists, can be used as described herein to provide painrelief

NTR agonists that can be used in combination with an opioid receptoragonist to treat pain include, without limitation, brain-penetratinganalogs of NT polypeptides. Such polypeptides can have amino acidsequences that are based on the sequence of NT(8-13) and can incorporateone or more amino acid analogs such as D- or L-neo-tryptophan (Fauq etal. (1998) Tetrahedron: Assymetry 9:4127-4134). Neo-tryptophan(2-amino-3-[1H-indolyl]propanoic acid) places the indole group oftryptophan in a unique orientation in terms of steric and electrostaticfields, such that polypeptides containing neo-tryptophan provide novelarrangements for side chain interactions. For example, this documentprovides methods for using polypeptides having the amino acid sequenceN-methyl-Arg-Lys-Pro-L-neo-Trp-tert-Leu-Leu (SEQ ID NO:3; referred toherein as NT69L). Examples of other polypeptides that are NT analogs areprovided herein in Table 1.

TABLE I Amino acid sequences of selected NTR agonists Polypeptide 1 2 34 5 6 7 8 9 10 11 12 13 NT p-Glu L-Leu L-Tyr L-Glu L-Asn L-Lys L-ProL-Arg L-Arg L-Pro L-Tyr L-Ile L-Leu NT(8-13) L-Arg L-Arg L-Pro L-TyrL-Ile L-Leu NT(9-13) L-Arg L-Pro L-Tyr L-Ile L-Leu NTW L-Arg L-Arg L-ProL-Trp L-Ile L-Leu NT (tert-Leu) L-Arg L-Arg L-Pro L-Tyr tert-Leu L-LeuEisai* N-methyl-Arg L-Lys L-Pro L-Trp tert-Leu L-Leu NT2 D-Lys L-ArgL-Pro L-Tyr L-Ile L-Leu NT24 “27” L-Arg D-Orn^(&) L-Pro L-Tyr L-IleL-Leu NT34 L-Arg L-Arg L-Pro L-3,1′-Nal^(#) L-Ile L-Leu NT64D L-ArgL-Arg L-Pro D-neo-Trp L-Ile L-Leu NT64L L-Arg L-Arg L-Pro L-neo-TrpL-Ile L-Leu NT65L L-Arg L-Arg L-Pro L-neo-Trp tert-Leu L-Leu NT66D D-LysL-Arg L-Pro D-neo-Trp tert-Leu L-Leu NT66L D-Lys L-Arg L-Pro L-neo-Trptert-Leu L-Leu NT67L D-Lys L-Arg L-Pro L-neo-Trp L-Ile L-Leu NT69LN-methyl-Arg L-Lys L-Pro L-neo-Trp tert-Leu L-Leu NT69L′ N-methyl-ArgL-Arg L-Pro L-neo-Trp tert-Leu L-Leu NT71 N-methyl-Arg DAB^($) L-ProL-neo-Trp tert-Leu L-Leu NT72 D-Lys L-Pro L-neo-Trp tert-Leu L-Leu NT73D-Lys L-Pro L-neo-Trp L-Ile L-Leu NT74 DAB L-Pro L-neo-Trp tert-LeuL-Leu NT75 DAB L-Pro L-neo-Trp L-Ile L-Leu NT76 L-Arg D-Orn L-ProL-neo-Trp L-Ile L-Leu NT77 L-Arg D-Orn L-Pro L-neo-Trp tert-Leu L-Leu*Tsuchiya Y et al., (1989) European Patent Application 89104302.8;^(#)naphthylalanine; ^($)diaminobutyric acid; ^(&)D-ornithine

As used herein, a “polypeptide” is any chain of amino acid residues,regardless of post-translational modification (e.g., phosphorylation orglycosylation). Polypeptides that can be used as NTR agonists typicallyare between 3 and 30 amino acids in length (e.g., 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 20, 25, or 30 amino acids in length). Forexample, a polypeptide can be between 3 and 13 amino acids in length.

The term “amino acid” refers to natural amino acids, unnatural aminoacids, and amino acid analogs, all in their D and L stereoisomers iftheir structures so allow. Natural amino acids include alanine (Ala),arginine (Arg), asparagine (Asn), aspartic acid (Asp), cysteine (Cys),glutamine (Gln), glutamic acid (Glu), glycine (Gly), histidine (His),isoleucine (Ile), leucine (Leu), lysine (Lys), methionine (Met),phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr),tryptophan (Trp), tyrosine (Tyr), and valine (Val). Unnatural aminoacids include, without limitation, azetidinecarboxylic acid,2-aminoadipic acid, 3-aminoadipic acid, beta-alanine, aminopropionicacid, 2-aminobutyric acid, 4-aminobutyric acid, 6-aminocaproic acid,2-aminoheptanoic acid, 2-aminoisobutyric acid, 3-aminoisobutyric acid,2-aminopimelic acid, 2,4-diaminoisobutyric acid, desmosine,2,2′-diaminopimelic acid, 2,3-diaminopropionic acid, N-ethylglycine,N-ethylasparagine, hydroxylysine, allo-hydroxylysine, 3-hydroxyproline,4-hydroxyproline, isodesmosine, allo-isoleucine, N-methylglycine,N-methylisoleucine, N-methylvaline, norvaline, norleucine, ornithine,pipecolic acid, N-methylarginine, 3,1-naphthylalanine,3,2-naphthylalanine, and neo-tryptophan.

An “analog” is a chemical compound that is structurally similar toanother but differs slightly in composition, as in the replacement ofone atom by an atom of a different element, the presence of a particularfunctional group, or the replacement of an amino acid with another aminoacid. An “amino acid analog” therefore is structurally similar to anaturally occurring amino acid molecule as is typically found in nativepolypeptides, but differs in composition such that either the C-terminalcarboxy group, the N-terminal amino group, or the side-chain functionalgroup has been chemically modified to another functional group. Aminoacid analogs include, without limitation, natural and unnatural aminoacids that are chemically blocked, reversibly or irreversibly, ormodified on their N-terminal amino group or their side-chain groups, andinclude, for example, methionine sulfoxide, methionine sulfone,S-(carboxymethyl)-cysteine, S-(carboxymethyl)-cysteine sulfoxide, andS-(carboxymethyl)-cysteine sulfone. Amino acid analogs may be naturallyoccurring, or can be synthetically prepared. Non-limiting examples ofamino acid analogs include, without limitation, asparticacid-(beta-methyl ester), an analog of aspartic acid; N-ethylglycine, ananalog of glycine; and alanine carboxamide, an analog of alanine. Otherexamples of amino acids and amino acids analogs are listed in Gross andMeienhofer, The Peptides: Analysis, Synthesis, Biology, Academic Press,Inc., New York (1983).

The stereochemistry of a polypeptide can be described in terms of thetopochemical arrangement of the side chains of the amino acid residuesabout the polypeptide backbone, which is defined by the peptide bondsbetween the amino acid residues and the I-carbon atoms of the bondedresidues. In addition, polypeptide backbones have distinct termini andthus direction. The majority of naturally occurring amino acids areL-amino acids. Naturally occurring polypeptides are largely comprised ofL-amino acids. D-amino acids are the enantiomers of L-amino acids andcan form “inverso” polypeptides (i.e., peptides corresponding to nativepeptides but made up of D-amino acids rather than L-amino acids).

Polypeptides can be modified for use in vivo by the addition, at theamino- or carboxy-terminal end, of a stabilizing agent to facilitatesurvival of the polypeptide in vivo. This can be useful in situations inwhich peptide termini tend to be degraded by proteases prior to cellularuptake. Such blocking agents can include, without limitation, additionalrelated or unrelated amino acid sequences that can be attached to theamino- and/or carboxy-terminal residues of a polypeptide (e.g., anacetyl group attached to the N-terminal amino acid or an amide groupattached to the C-terminal amino acid). Such attachment can be achievedeither chemically, during the synthesis of the polypeptide, or byrecombinant DNA technology using methods familiar to those of ordinaryskill in the art. In some cases, blocking agents such as pyroglutamicacid or other molecules known in the art can be attached to the amino-and/or carboxy-terminal residues, or the amino group at the aminoterminus or the carboxy group at the carboxy terminus can be replacedwith a different moiety.

Polypeptides also can contain an amino acid tag. A “tag” is generally ashort amino acid sequence that provides a ready means of detection orpurification through interactions with an antibody against the tag orthrough other compounds or molecules that recognize the tag. Forexample, tags such as c-myc, hemagglutinin, polyhistidine, or Flag® canbe used to aid purification and detection of a polypeptide. As anexample, a polypeptide with a polyhistidine tag can be purified based onthe affinity of histidine residues for nickel ions (e.g., on a Ni-NTAcolumn), and can be detected in western blots by an antibody againstpolyhistidine (e.g., the Penta-His antibody; Qiagen, Valencia, Calif.).Tags can be inserted anywhere within a polypeptide sequence, includingat the amino- or carboxy-terminus.

NTR agonists that can be used as described herein also can bepeptidomimetic compounds designed on the basis of the amino acidsequences of NT polypeptides. Peptidomimetic compounds are synthetic,non-peptide compounds having a three-dimensional conformation (i.e., a“peptide motif”) that is substantially the same as the three-dimensionalconformation of a selected polypeptide, and thus can confer the same orsimilar function as the selected polypeptide. Peptidomimetic compoundscan be designed to mimic any of the NT polypeptides provided herein.

In some cases, a peptidomimetic compound can be protease resistant.Furthermore, peptidomimetic compounds may have additionalcharacteristics that enhance therapeutic effects, such as increased cellpermeability and prolonged biological half-life. Such compoundstypically have a backbone that is partially or completely non-peptide,but with side groups that are identical or similar to the side groups ofthe amino acid residues that occur in the polypeptide upon which thepeptidomimetic compound is based. Several types of chemical bonds (e.g.,ester, thioester, thioamide, retroamide, reduced carbonyl, dimethyleneand ketomethylene) can be used as substitutes for peptide bonds in theconstruction of peptidomimetic compounds.

Polypeptides that can be used as described herein can be produced by anumber of methods, many of which are well known in the art. By way ofexample and not limitation, a polypeptide can be obtained by extractionfrom a natural source (e.g., from isolated cells, tissues, or bodilyfluids), by expression of a recombinant nucleic acid encoding thepolypeptide (as, for example, described herein), or by chemicalsynthesis (e.g., by solid-phase synthesis or other methods well known inthe art, including synthesis with an ABI peptide synthesizer; AppliedBiosystems, Foster City, Calif.).

NT analogs such as NT69L can be synthesized using Fmoc chemistry witht-butyl-protected side chains on an automated peptide synthesizer, forexample. See, U.S. Pat. No. 6,214,790 and Cusack et al. (2000) BrainRes. 856:48-54. NT69L has the amino acid sequenceN-methyl-Arg-Lys-Pro-L-neo-Trp-tert-Leu-Leu (SEQ ID NO:3). The L-neo-Trpresidue can be synthesized by, for example, the method of Fauq et al.(Fauq et al. supra), or by methods disclosed in U.S. Pat. No. 6,214,790.Once synthesized, the polypeptide can be purified by, for example, HPLC(e.g., reverse phase HPLC).

NT polypeptides also can be prepared by recombinant technology usingisolated nucleic acid molecules encoding the polypeptides. As usedherein, “nucleic acid” refers to both RNA and DNA, including cDNA,genomic DNA, and synthetic (e.g., chemically synthesized) DNA. Thenucleic acid can be double-stranded or single-stranded (i.e., a sense oran antisense single strand).

Nucleic acids encoding NT polypeptides can be contained within nucleicacid vectors. A vector is a replicon, such as a plasmid, phage, orcosmid, into which another nucleic acid segment may be inserted so as tobring about the replication of the inserted segment. Vectors that areuseful to produce NT polypeptides typically are expression vectors, inwhich the nucleotides encode an NT polypeptide with an initiatormethionine, operably linked to expression control sequences. As usedherein, “operably linked” means incorporated into a genetic construct sothat expression control sequences effectively control expression of acoding sequence of interest. An “expression control sequence” is anucleic acid sequence that controls and/or regulates the transcriptionand translation of another nucleic acid sequence, and an “expressionvector” is a vector that includes expression control sequences, so thata relevant nucleic acid segment incorporated into the vector istranscribed and translated. A coding sequence is “operably linked” and“under the control” of transcriptional and translational controlsequences in a cell when a polymerase transcribes the coding sequenceinto mRNA, which then is translated into the polypeptide encoded by thecoding sequence.

Methods well known to those skilled in the art may be used to subcloneisolated nucleic acid molecules encoding NT polypeptides into expressionvectors containing relevant coding sequences and appropriatetranscriptional/translational control signals. See, for example,Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd edition),Cold Spring Harbor Laboratory, New York (1989); and Ausuble et al.,Current Protocols in Molecular Biology, Green Publishing Associates andWiley Interscience, New York (1989). Expression vectors can be used toproduce NT polypeptides in a variety of systems (e.g., bacteria, yeast,insect cells, and mammalian cells). Examples of suitable expressionvectors include, without limitation, plasmids and viral vectors derivedfrom herpes viruses, retroviruses, vaccinia viruses, adenoviruses, andadeno-associated viruses. A wide variety of expression vectors andsystems are commercially available, including the pET series ofbacterial expression vectors (Novagen, Madison, Wis.), the Adeno-Xexpression system (Clontech), the Baculogold baculovirus expressionsystem (BD Biosciences Pharmingen, San Diego, Calif.), and the pCMV-Tagvectors (Stratagene, La Jolla, Calif.).

Expression systems that can be used for small or large scale productionof NT polypeptides include, without limitation, microorganisms such asbacteria (e.g., E. coli and B. subtilis) transformed with recombinantbacteriophage DNA, plasmid DNA, or cosmid DNA expression vectorscontaining nucleic acid molecules encoding NT polypeptides; yeast (e.g.,S. cerevisiae) transformed with recombinant yeast expression vectorscontaining nucleic acid molecules encoding NT polypeptides; insect cellsystems infected with recombinant virus expression vectors (e.g.,baculovirus) containing nucleic acid molecules encoding NT polypeptides;plant cell systems infected with recombinant virus expression vectors(e.g., tobacco mosaic virus) or transformed with recombinant plasmidexpression vectors (e.g., Ti plasmid) containing nucleic acid moleculesencoding NT polypeptides; or mammalian cell systems (e.g., primary cellsor immortalized cell lines such as COS cells, CHO cells, HeLa cells, HEK293 cells, and 3T3 L1 cells) harboring recombinant expression constructscontaining promoters derived from the genome of mammalian cells (e.g.,the metallothionein promoter) or from mammalian viruses (e.g., theadenovirus late promoter and the cytomegalovirus promoter), along withnucleic acid molecules encoding NT polypeptides.

NT polypeptides can be substantially pure NT polypeptides. The term“substantially pure” as used herein with reference to a polypeptidemeans the polypeptide is substantially separated from other moleculesand compounds. Thus, a naturally occurring polypeptide that issubstantially pure is substantially free of other polypeptides, lipids,carbohydrates, and nucleic acid with which it associates in nature. Anon-naturally occurring polypeptide (e.g., a synthetic polypeptide or apeptidomimetic) that is substantially pure is substantially free of thechemical components included in the synthesis reaction. Typically, asubstantially pure polypeptide will yield a single major band on anon-reducing polyacrylamide gel. A substantially pure NT polypeptide canbe at least about 60 percent pure (e.g., at least about 65, 70, 75, 80,85, 90, 95, or 99 percent pure). It is understood that an NT polypeptideis considered substantially pure if it has been purified and then mixedwith, for example, an opioid receptor agonist (e.g., morphine), anadjuvant, or a pharmaceutical carrier, as the NT polypeptide isseparated from the cellular components with which it is associated innature or separated from the components with which it is associated in asynthesis reaction. Suitable methods for purifying NT polypeptides caninclude, for example, affinity chromatography, immunoprecipitation, sizeexclusion chromatography, and ion exchange chromatography. The extent ofpurification can be measured by any appropriate method, including butnot limited to: column chromatography, polyacrylamide gelelectrophoresis, or high-performance liquid chromatography.

Small molecules also can be used in the methods provided herein to treatpain. Such small molecules (i.e., non-polypeptide NTR agonists) can beisolated and identified using assays such as ELISA and binding assays(e.g., affinity chromatography). For example, NTR molecules can becoated in the wells of a microtiter plate or coupled to a chromatographyresin. Cellular extracts or solutions containing a cocktail of smallmolecules can be incubated in the wells or with the resin. Moleculesthat do not bind can be washed away, while bound molecules can be elutedby, for example, washing with a buffer containing a relatively highconcentration of salt.

Compositions

NTR agonists and opioid receptor agonists can be incorporated intocompositions that can be used to treat pain. Any method for formulatingand subsequently administering such compositions can be used. Dosinggenerally is dependent on the severity and location of the pain, withthe course of treatment lasting from several days to several months, oruntil the underlying source of the pain (e.g., wound or disease) isalleviated or removed. Optimum dosages can vary depending on therelative potency of individual NTR agonists and opioid receptoragonists, and generally can be estimated based on EC₅₀ found to beeffective in in vitro and in vivo animal models. Typically, dosage isfrom 0.01 μg to 100 g per kg of body weight. Compositions containing NTRagonists and opioid receptor agonists can be given once or more daily,weekly, or even less often.

The methods provided herein include the administration of pharmaceuticalcompositions and formulations that include an NTR agonist and an opioidreceptor agonist. A composition containing at least one NTR agonist andat least one opioid receptor agonist can be admixed, encapsulated,conjugated or otherwise associated with other molecules, molecularstructures, or mixtures of molecules such as, for example, liposomes,receptor targeted molecules, or oral, rectal, topical or otherformulations, for assisting in uptake, distribution and/or absorption.

A “pharmaceutically acceptable carrier” is a pharmaceutically acceptablesolvent, suspending agent, or any other pharmacologically inert vehiclefor delivering one or more therapeutic compounds (e.g., NT69L andmorphine) to a subject. Pharmaceutically acceptable carriers can beliquid or solid, and can be selected with the planned manner ofadministration in mind so as to provide for the desired bulk,consistency, and other pertinent transport and chemical properties, whencombined with one or more therapeutic compounds and any other componentsof a given pharmaceutical composition. Typical pharmaceuticallyacceptable carriers include, by way of example and not limitation:water; saline solution; binding agents (e.g., polyvinylpyrrolidone orhydroxypropyl methylcellulose); fillers (e.g., lactose and other sugars,gelatin, or calcium sulfate); lubricants (e.g., starch, polyethyleneglycol, or sodium acetate); disintegrates (e.g., starch or sodium starchglycolate); and wetting agents (e.g., sodium lauryl sulfate).

Pharmaceutical compositions can be administered by a number of methods.Administration can be, for example, topical (e.g., transdermal,ophthalmic, or intranasal); pulmonary (e.g., by inhalation orinsufflation of powders or aerosols); oral; or parenteral (e.g., bysubcutaneous, intrathecal, intraventricular, intramuscular, orintraperitoneal injection, or by intravenous drip). Administration canbe rapid (e.g., by injection) or can occur over a period of time (e.g.,by slow infusion or administration of slow release formulations). Fortreating tissues in the central nervous system, an NTR agonist and anopioid receptor agonist can be administered by injection or infusioninto the cerebrospinal fluid, preferably with one or more agents capableof promoting penetration of the agonists across the blood-brain barrier.

Formulations for topical administration of NTR agonists and opioidreceptor agonists include, for example, sterile and non-sterile aqueoussolutions, non-aqueous solutions in common solvents such as alcohols, orsolutions in liquid or solid oil bases. Such solutions also can containbuffers, diluents and other suitable additives. Pharmaceuticalcompositions and formulations for topical administration can includetransdermal patches, ointments, lotions, creams, gels, drops,suppositories, sprays, liquids, and powders. Conventional pharmaceuticalcarriers, aqueous, powder or oily bases, thickeners and the like may benecessary or desirable.

Compositions and formulations for oral administration of NTR agonistsand opioid receptor agonists include, for example, powders or granules,suspensions or solutions in water or non-aqueous media, capsules,sachets, or tablets. Such compositions also can incorporate thickeners,flavoring agents, diluents, emulsifiers, dispersing aids, or binders.

Compositions and formulations for parenteral, intrathecal orintraventricular administration can include sterile aqueous solutions,which also can contain buffers, diluents and other suitable additives(e.g., penetration enhancers, carrier compounds and otherpharmaceutically acceptable carriers).

Pharmaceutical compositions include, but are not limited to, solutions,emulsions, aqueous suspensions, and liposome-containing formulations.These compositions can be generated from a variety of components thatinclude, for example, preformed liquids, self-emulsifying solids andself-emulsifying semisolids. Emulsions are often biphasic systemscomprising of two immiscible liquid phases intimately mixed anddispersed with each other; in general, emulsions are either of thewater-in-oil (w/o) or oil-in-water (o/w) variety. Emulsion formulationshave been widely used for oral delivery of therapeutics due to theirease of formulation and efficacy of solubilization, absorption, andbioavailability.

Liposomes are vesicles that have a membrane formed from a lipophilicmaterial and an aqueous interior that can contain a composition providedherein. Liposomes can be particularly useful due to their specificityand the duration of action they offer from the standpoint of drugdelivery. Liposome compositions can be formed from phosphatidylcholine,dimyristoyl phosphatidylcholine, dipalmitoyl phosphatidylcholine,dimyristoyl phosphatidylglycerol, or dioleoyl phosphatidylethanolamine,for example. Numerous lipophilic agents are commercially available,including Lipofectin® (Invitrogen/Life Technologies, Carlsbad, Calif.)and Effectene3 (Qiagen, Valencia, Calif.).

Compositions containing an NTR agonist and an opioid receptor agonistcan further encompass any pharmaceutically acceptable salts, esters, orsalts of such esters, or any other compound which, upon administrationto a mammal (e.g., a human), is capable of directly or indirectlyproviding the biologically active agonist or residue thereof.Accordingly, for example, pharmaceutically acceptable salts of an NTRagonist or an opioid receptor agonist, prodrugs of an NTR agonist or anopioid receptor agonist, pharmaceutically acceptable salts of suchprodrugs, and other bioequivalents can be used as described herein. Aprodrug is a therapeutic agent that is prepared in an inactive form andis converted to an active form (i.e., drug) within the body or cellsthereof by the action of endogenous enzymes or other chemicals and/orconditions. A pharmaceutically acceptable salt of an NTR agonist or anopioid receptor agonist can be a salt that retains the desiredbiological activity of the parent agonist molecule without impartingundesired toxicological effects. Examples of pharmaceutically acceptablesalts include, but are not limited to, salts formed with cations (e.g.,sodium, potassium, calcium, or polyamines such as spermine); acidaddition salts formed with inorganic acids (e.g., hydrochloric acid,hydrobromic acid, sulfuric acid, phosphoric acid, or nitric acid); saltsformed with organic acids (e.g., acetic acid, citric acid, oxalic acid,palmitic acid, or fumaric acid); and salts formed from elemental anions(e.g., chlorine, bromine, and iodine).

Pharmaceutical compositions containing an NTR agonist and an opioidreceptor agonist also can incorporate penetration enhancers that promotethe efficient delivery of, for example, polypeptides, small molecules,or other molecules, to the skin of animals. Penetration enhancers canenhance the diffusion of both lipophilic and non-lipophilic drugs acrosscell membranes. Penetration enhancers can be classified as belonging toone of five broad categories, i.e., surfactants (e.g., sodium laurylsulfate, polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetylether); fatty acids (e.g., oleic acid, lauric acid, myristic acid,palmitic acid, and stearic acid); bile salts (e.g., cholic acid,dehydrocholic acid, and deoxycholic acid); chelating agents (e.g.,disodium ethylenediaminetetraacetate, citric acid, and salicylates); andnon-chelating non-surfactants (e.g., unsaturated cyclic ureas).

Compositions provided herein can contain other adjunct componentsconventionally found in pharmaceutical compositions. Thus, thecompositions provided herein can include compatible, pharmaceuticallyactive materials such as, for example, antipruritics, astringents, localanesthetics, or anti-inflammatory agents, or additional materials usefulin physically formulating various dosage forms such as dyes, flavoringagents, preservatives, antioxidants, opacifiers, thickening agents, orstabilizers. Furthermore, a composition provided herein can be mixedwith auxiliary agents, e.g., lubricants, preservatives, stabilizers,wetting agents, emulsifiers, salts for influencing osmotic pressure,buffers, colorings, flavorings, and aromatic substances. When added,however, such materials should not unduly interfere with the biologicalactivities of the NTR agonists or opioid receptor agonists within thecompositions. The formulations can be mixed with auxiliary agents (e.g.,lubricants, preservatives, stabilizers, wetting agents, emulsifiers,salts for influencing osmotic pressure, buffers, colorings, flavoringsand/or aromatic substances and the like) that do not deleteriouslyinteract with the agonists of the formulation.

Pharmaceutical formulations can be presented conveniently in unit dosageform, and can be prepared according to conventional techniques wellknown in the pharmaceutical industry. Such techniques include the stepof bringing into association the active ingredients (e.g., NT69L andmorphine) with the desired pharmaceutical carrier(s). Typically, aformulation can be prepared by uniformly bringing the active ingredientsinto intimate association with liquid carriers or finely divided solidcarriers or both, and then, if necessary, shaping the product.Formulations can be sterilized if desired, provided that the method ofsterilization does not interfere with the effectiveness of the agonistscontained in the formulation.

Compositions containing an NTR agonist and an opioid receptor agonistcan be formulated into any of many possible dosage forms such as, butnot limited to, tablets, capsules, liquid syrups, soft gels,suppositories, and enemas. Compositions also can be formulated assuspensions in aqueous, non-aqueous or mixed media. Aqueous suspensionsfurther can contain substances that increase the viscosity of thesuspension including, for example, sodium carboxymethylcellulose,sorbitol, and/or dextran. Suspensions also can contain stabilizers.

The synergistic combination can be delivered transdermally, by means ofa transdermal patch for systemic effect, orally or as an intranasalspray for systemic effect, as a suppository, intravenously,intramuscularly or subcutaneously, or as a topical application (cream orpatch), etc. When administered orally, in a “sustained release”formulation, the NTR agonist can be on the outside and the opioidreceptor agonist (e.g., an opiate) on the inside. This can allow the NTRagonist to be released first, and the opioid receptor agonist later. Thedopaminergic blockade caused by the NTR agonist can therefore diminishor eliminate the reward system and so addictive behaviour. The reverseformulation, with the opioid receptor agonist on the outside, however,can prevent a polypeptide NTR agonist peptide from degradation by gutpeptidases.

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

EXAMPLES

Examples of neurotensin analogs and neurotensin receptor agonists, eachwith neotryptophan, include those listed in the following tables. Thetable immediately below lists neurotensin analogs that includeneo-tryptophan.

NT64L [L-neo-Trp¹¹]NT(8-13) NT72D[D-Lys⁹,D-neo-Trp¹¹,tert-Leu¹²]NT(9-13) NT64D [D-neo-Trp¹¹]NT(8-13)NT73L [D-Lys⁹,L-neo-Trp¹¹]NT(9-13) NT65L [L-neo-Trp¹¹,tert-Leu¹²]NT(8-13) NT73D [D-Lys⁹,D-neo-Trp¹¹]NT(9-13) NT65D[D-neo-Trp¹¹, tert-Leu¹²]NT(8-13) NT74L[DAB⁹,L-neo-Trp¹¹,tert-Leu¹²]NT(9-13) NT66L [D-Lys⁸, L-neo-Trp¹¹,tert-Leu¹²]NT(8-13) NT74D [DAB⁹,Pro,D-neo-Trp¹¹,tert-Leu¹²]NT(9-13)NT66D [D-Lys⁸, D-neo-Trp¹¹, tert-Leu¹²]NT(8-13) NT75L[DAB⁸,L-neo-Trp¹¹]NT(8-13) NT67L [D-Lys⁸, L-neo-Trp¹¹]NT(8-13) NT75D[DAB⁸,D-neo-Trp¹¹]NT(8-13) NT67D [D-Lys⁸, D-neo-Trp¹¹]NT(8-13) NT76L[D-Orn⁹,L-neo-Trp¹¹]NT(8-13) NT69L[N-methyl-Arg⁸,L-Lys⁹,L-neo-Trp¹¹,tert- NT76D[D-Orn⁹,D-neo-Trp¹¹]NT(8-13) Leu¹²]NT(8-13) NT69D[N-methyl-Arg⁸,L-Lys⁹,D-neo-Trp¹¹,tert- NT77L[D-Orn⁹,L-neo-Trp¹¹,tert-Leu¹²]NT(8-13) Leu¹²]NT(8-13) NT71L[N-methyl-Arg⁸,DAB⁹,L-neo-Trp¹¹,tert- NT77D[D-Orn⁹,D-neo-Trp¹¹,tert-Leu¹²]NT(8-13) leu¹²]NT(8-13) NT71D[N-methyl-Arg⁸,DAB⁹,D-neo-Trp¹¹,tert- NT78L[N-methyl-,D-Orn⁹,L-neo-Trp¹¹,tert- leu¹²]NT(8-13) Leu¹²]NT(8-13) NT72L[D-Lys⁹,L-neo-Trp¹¹,tert-Leu¹²]NT(9-13) NT78D[N-methyl-Arg⁸,D-Orn⁹,D-neo-Trp¹¹,tert- Leu¹²]NT(8-13)

TABLE 1 Amino Acid Sequence and Analytical Data for NT, NT|8-13|, andKK1-19 amino acid sequence MW (g/mol) peptide 1 2 3 4 5 6 7 8 9 10 11 1213 obsd^(a) (calcd)^(b) NT Glu L-Leu L-Tyr L-Glu L-Asn L-Lys L-Pro L-ArgL-Arg L-Pro L-Tyr L-Ile L-Leu NA^(c) NT[8-13] — — — — — — — L-Arg L-ArgL-Pro L-Tyr L-Ile L-Leu NA KK1 — — — — — — — N₃-L-Hlys L-Arg L-Pro L-TyrL-Ile L-Leu 829.5 (829.0) KK2 — — — — — — — N₃-1^(d) L-Arg L-Pro L-TyrL-Ile L-Leu 843.5 (843.0) KK3 — — — — — — — N₃-2 L-Arg L-Pro L-Tyr L-IleL-Leu 857.5 (857.1) KK4 — — — — — — — N₃-3 L-Arg L-Pro L-Tyr L-Ile L-Leu871.5 (872.1) KK5 — — — — — — — N₃-L-Lys L-Arg L-Pro L-Tyr L-Ile L-Leu815.5 (815.0) KK6 — — — — — — — N₃-4 L-Arg L-Pro L-Tyr L-Ile L-Leu 829.6(829.0) KK7 — — — — — — — N₃-5 L-Arg L-Pro L-Tyr L-Ile L-Leu 843.5(843.0) KK8 — — — — — — — N₃-6 L-Arg L-Pro L-Tyr L-Ile L-Leu 857.6(858.1) KK9 — — — — — — — N₃-L-Orn L-Arg L-Pro L-Tyr L-Ile L-Leu 801.4(801.0) KK10 — — — — — — — N₃-7 L-Arg L-Pro L-Tyr L-Ile L-Leu 815.5(815.0) KK11 — — — — — — — N₃-8 L-Arg L-Pro L-Tyr L-Ile L-Leu 829.5(829.0) KK12 — — — — — — — N₃-9 L-Arg L-Pro L-Tyr L-Ile L-Leu 843.5(844.0) KK13 — — — — — — — N₃-L-Hlys L-Arg L-Pro L-Tyr L-tert-Leu L-Leu829.5 (829.0) KK14 — — — — — — — N₃-L-Hlys L-Arg L-Pro L-Trp L-tert-LeuL-Leu 852.5 (852.0) KK15 — — — — — — — N₃-L-Arg L-Arg L-Pro L-Tyr L-IleL-Leu 843.5 (843.0) KK16 — — — — — — — 10 L-Arg L-Pro L-Tyr L-Ile L-Leu831.6 (831.0) KK17 — — — — — — — 11 L-Arg L-Pro L-Tyr L-Ile L-Leu 845.6(845.0) KK18 — — — — — — — 12 L-Arg L-Pro L-Tyr L-Ile L-Leu 843.6(813.0) KK19 — — — — — — — 13 L-Arg L-Pro L-Tyr L-Ile L-Leu 843.6(843.0)See Kokko, J. Med. Chem., (2003) 46:4141-4148, incorporated herein byreference.

EXAMPLE 1 Treating Pain Using A Combination of an NTR Agonist (NT69L)and an Opioid Receptor Agonist

Sprague Dawley male rats (150-250 g) were housed in a temperaturecontrolled room with a 12:12 hour light/dark cycle and were givenstandard rat chow and water ad lib. Animals were injected with eitherNT69L or morphine or the combination of the two drugs in separateinjections, one immediately after the other. All drugs were injectedintraperitoneally. Fifteen minutes after the injection, animals weretested on the hot plate (time=0 on the graph of FIG. 1). The baselinehot plate data were obtained immediately prior to the experiment. Thehot plate methods are described elsewhere (Tyler et al., Brain Research,792:246-52 (1998)). Briefly, the hot plate was performed to determinepain sensitivity. The rats were placed on a metal plate (15×20 cm),maintained at a temperature of 52.5±0.15° C. The latency between thetime the rat is placed on the surface and the time it licks either ofits hind paws was measured. Failure to respond in 30 sec resulted inending the trial and assignment of that latency. Hot plate tests werescored as the percent of Maximum Possible Effect (% MPE) and calculatedaccording to the following equation: % MPE=[(post-drug latency−pre-druglatency)/(cut-off−pre-drug latency)]×100; where 30 second is thecut-off.

Animals treated with both NT69L and morphine exhibited anantinociceptive effect that was greater than the sum of the effects ofeach compound separately (FIG. 1). These results indicate that thecombination of an NTR agonist and an opioid receptor agonist provide alevel of pain relief that is greater than that observed with either anNTR agonist or an opioid receptor agonist alone.

EXAMPLE 2 Treating Pain Using a Combination of an NTR Agonist (NT72 orNT77) and an Opioid Receptor Agonist

As in Example 1, Sprague Dawley male rats (150-250 g) were housed in atemperature controlled room with a 12:12 hour light/dark cycle and weregiven standard rat chow and water ad lib. Animals were injected witheither NT77, NT72, morphine, or the combination of morphine with eitherNT77 or NT72 in separate injections, one immediately after the other.All drugs were injected intraperitoneally. Fifteen minutes after theinjection, animals were tested on the hot plate (time=0 on the graph ofFIG. 2). The baseline hot plate data were obtained immediately prior tothe experiment. The hot plate methods are described elsewhere (Tyler etal., Brain Research, 792:246-52 (1998)). Briefly, the hot plate wasperformed to determine pain sensitivity. The rats were placed on a metalplate (15×20 cm), maintained at a temperature of 52.5±0.15° C. Thelatency between the time the rat is placed on the surface and the timeit licks either of its hind paws was measured. Failure to respond in 30sec resulted in ending the trial and assignment of that latency. Hotplate tests were scored as the percent of Maximum Possible Effect (%MPE) and calculated according to the following equation: %MPE=[(post-drug latency−pre-drug latency)/(cut-off−pre-druglatency)]×100; where 30 second is the cut-off.

Animals treated with both NT77 and morphine or NT72 and morphineexhibited an antinociceptive effect that was greater than the sum of theeffects of each compound separately (FIG. 2). These results indicatethat the combination of an NTR agonist and an opioid receptor agonistprovide a level of pain relief that is greater than that observed witheither an NTR agonist or an opioid receptor agonist alone.

EXAMPLE 3

Male CD-1 mice (25-30 g) are used in each example. Drugs areadministered systemically via subcutaneous or intraperitonealinjections. Gastrointestinal transit is assessed by measuring thedistance traveled by a charcoal meal (Paul and Pasternak 1988),incorporated herein by reference.

Analgesia is assessed 30 minutes post-injection using the radiant heattail-flick assay. Baseline latencies range between 2.0 and 3.2 seconds.A maximal cutoff latency of 10 seconds is set to minimize tissue damage.Analgesia is assessed quantally as a doubling or greater of the baselinelatency for each mouse. Quantal measures have long been used in thisassay (D'Amour and Smith, 1941; Le Bars et al., 2001), as previouslypublished by Pasternak et al, 19801a,b; Rossi et al, 1995, 1996; Neilanet al., 2001). Groups of mice are compared using Fisher's extract test.ED50 values and 95% confidence limits are calculated by probit analysis(Tallarida, 2000).

To assess the statistical significance of the combinations, completedose-response data are determined for each compound and are examinedwith probit regression analysis with the aid of Pharm tools Pro (McCaryGroup, Elkins Park, Pa.). Each opiate compound is paired in afixed-ratio combination with the neurotensin in question (NT69L orother) to assess whether the combination displays enhanced potencyversus opiate alone indicative of synergism. That assessment is made bydetermining the composite line of additivity for the combination andcomparing that line to the dose-response regression line of theexperimentally determined combination using ANOVA (Tallarida, 2000). Agraphical assessment of synergy is also presented using isobolographicanalysis (Roerig et al., 1984; Kolesnikov et al., 1996, 2000; Tallaridaand Raffa, 1996; Tallarida et al., 1997).

Although the foregoing invention has, for the purposes of clarity andunderstanding, been described in some detail by way of illustration andexample, it will be obvious that certain changes and modifications maybe practiced which will still fall within the scope of the appendedclaims. It will also be understood that any feature or features from anyone embodiment, or any reference cited herein, may be used with anycombination of features from any other embodiment.

OTHER EMBODIMENTS

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims. It is to be understood that any feature or aspect ofany composition, formulation, combination, or method described hereincan be used together with any other composition, formulation,combination, or method described herein.

What is claimed is:
 1. A method for treating pain, said methodcomprising administering a neurotensin receptor agonist and morphine toa mammal in need thereof, wherein the neurotensin receptor agonist is apolypeptide having an N-methyl-Arginine at position 8, an L-Proline atposition 10, a tert-Leucine at position 12, and an L-Leucine at position13, based on the numbering of NT(1-13).
 2. The method of claim 1,wherein the neurotensin receptor agonist is NT69L.
 3. The method ofclaim 1, wherein the neurotensin receptor agonist is NT69L′.
 4. Themethod of claim 1, wherein the neurotensin receptor agonist containsL-Arginine at position
 9. 5. A method for treating pain, said methodcomprising administering a neurotensin receptor agonist and morphine toa mammal in need thereof, wherein the neurotensin receptor agonist is apolypeptide having an Arginine at position 8, a Proline at position 10,and a Leucine or Isoleucine at position 12, and a Leucine at position13, based on the numbering of NT(1-13).
 6. The method of claim 5,wherein the Arginine at position 8 is an N-methyl-Arginine.
 7. Themethod of claim 5, wherein the Arginine at position 8 is an L-Arginine.8. The method of claim 5, wherein the Proline at position 10 is anL-Proline.
 9. The method of claim 5, wherein the Leucine or Isoleucineat position 12 is an L-Isoleucine.
 10. The method of claim 5, whereinthe Leucine or Isoleucine at position 12 is a tert-Leucine.
 11. Themethod of claim 5, wherein the Leucine at position 13 is an L-Leucine.12. The method of claim 5, wherein the neurotensin receptor agonistcontains L-Arginine at position 9
 13. The method of claim 5, wherein theneurotensin receptor agonist is NT69L.
 14. The method of claim 5,wherein the neurotensin receptor agonist is NT69L′.
 15. The method ofclaim 5, wherein the neurotensin receptor agonist is NT.
 16. The methodof claim 5, wherein the neurotensin receptor agonist is NT(8-13). 17.The method of claim 5, wherein the neurotensin receptor agonist is NT76.